JP5241517B2 - Microneedle device for allergy diagnosis - Google Patents

Description

  The present invention relates to a microneedle device for allergy diagnosis for performing simple, rapid and clear skin allergy diagnosis.

  Immunity is a mechanism to prevent illness from occurring originally, but the immune reaction may cause a bad reaction to the body. This is called allergy, and the origin of the reaction is the allergic antigen ( Antigen).

The contact allergy test is generally called a patch test. In such a contact allergy test, a contact allergy test sheet in which an antigen is placed on the surface of the sheet and can be applied to the skin of a subject is used. In general, a sheet is attached to the skin of the subject, peeled off from the skin after a certain contact time has elapsed, and the presence or degree of allergy is determined with the naked eye based on changes in the color of the skin.

  The patch test here refers to a diagnostic method that can be easily performed without bleeding or pain regardless of whether or not the skin epidermis is damaged.

The skin is composed of the outermost stratum corneum, epidermis, dermis, and subcutaneous connective tissue. Usually, the stratum corneum consisting of a dead cell layer and a lipid bilayer exhibits a strong barrier function against many substances. In the epidermis layer, antigen-presenting cells called Langerhans cells exist and have an immune function. Langerhans cells capture protein antigens that have entered the skin, break down inside, and express peptide fragments on MHC molecules. The MHC-peptide complex moves from the imported lymphatic vessel to the subcortical layer of the regional lymph node, and contacts through the T cell and the finger process cell. Langerhans cells, by moving in this manner, is transmitted to the T _H cells antigen is present in the lymph nodes from the skin. Langerhans cells have MHC class II molecules necessary to present antigen to the T _H cells.

  As described above, the contact time of the patch test is normally 48 hours from the viewpoint of taking a sufficient time because of the strong barrier function by the stratum corneum of the skin. Further, in the conventional contact allergy test, since the change in skin color is determined with the naked eye, skill is required for the determination and there is a problem in accuracy in determining the degree of allergy.

  In addition, since antigens are often high molecular weight substances such as proteins, absorption is difficult, and recently, iontophoresis and electroporation as disclosed in Patent Document 1 and Patent Document 2 have been proposed as means for enhancing antigen permeation. Studies on external dosage forms using a device are also underway. However, these have never been used directly for allergy patch tests, and no examples have been used for contact allergy tests. In other words, the use of iontophoresis and electroporation devices for contact allergy testing has been apt to be avoided for reasons of convenience, simplicity, rapidity, and skin irritation.

  Furthermore, recently, various microneedles and accompanying coating techniques have been developed. Patent Document 3 discloses an interface including a microneedle in which a skin piercing member holds a coating as a device for inoculating a vaccine through the skin. However, there is no example used for the contact allergy test here.

  By the way, in order to solve the problems of long-time contact by the patch test due to the strong barrier function by the stratum corneum of the skin and the unclearness of the color change of the skin, the antigen is directly administered subcutaneously with an injection needle or needle. However, due to the nature of allergy diagnosis, it is necessary to diagnose various antibodies, and it is clear that there are problems when considering the burden on the subject.

  In addition, the diagnosis of drug allergy to antibiotics, diabetes, and infectious diseases (tuberculosis, hepatitis, HIV, etc.) has been devised by the skin reaction test, which is useful in the skin reaction test by prick test or intradermal administration test Sex has been confirmed. The prick test is a method of holding an intradermal needle in a horizontal direction with respect to the skin and puncturing the skin to the extent that it does not bleed, and is a method of diagnosing immediate drug allergy. Although this method is safer than the intradermal administration test, the procedure is cumbersome and time consuming, requires the skill of a health care professional, and has the risk of bleeding depending on the degree of puncture of the needle into the skin. Special attention should be given to patients who may be infected with infectious diseases. In addition, in the intradermal reaction test, in addition to requiring administration techniques for direct injection into the skin, causing problems such as pain, hemorrhage, and excessive reaction has caused problems. The reaction test has been abolished.

For example, in Patent Document 4, a test in which one or a plurality of protruding lancet blade needles that can pierce the skin up to a certain limit when allergic substances are applied or adhered to a part of the adhesive surface of the adhesive tape piece is held. Although a piece is disclosed, the length of the lancet is in the range of 0.762 mm to 1.524 mm, and administration to the dermis past the epidermis where there are clearly many antigen presenting cells that are responsible for immune function. Yes, as with the prick test, it left many problems in terms of burden on the subject due to pain and efficient allergy diagnosis.
Japanese Patent No. 2506543 Special table 2002-535100 gazette Special table 2004-528900 gazette JP 58-131919 A

As mentioned above, the conventional patch test usually takes 48 hours, and it cannot be called a simple and safe diagnostic method in the skin prick test or the intradermal administration test, and the skin color change is unclear. There were many. Furthermore, the material of the microneedle has so far been often a metal as disclosed in Patent Document 3, but in the allergy diagnosis, there are many cases where the metal itself acts as an antigen and cannot be accurately diagnosed. . In addition, it is conceivable to use a metal material that does not easily act as an allergic antigen such as titanium. However, since the material is expensive, a microneedle that is not practical for use as a diagnosis has been demanded. Further, the lancet shown in Patent Document 4 has a length of 0.762 mm to 1.524 mm, which is longer than that of the microneedle, and there is a problem of generation of pain and bleeding in addition to the inability to make an efficient determination.
An object of the present invention is to provide an inexpensive microneedle device for allergy diagnosis that can perform a clear skin determination in a short time with a simple operation in allergy diagnosis.

  As a result of examining the transdermal administration method of allergic antigens using immunized rats, the highest allergic reaction was shown by using microneedles and coating the microneedles. Furthermore, the inventors have found that rapid and accurate diagnosis is possible by using a non-metallic synthetic or natural resin material as the material of the microneedle, and completed the present invention.

That is, the object is to provide a substrate, a length of 50 μm to 500 μm that can be pierced on the substrate, and a density of 100 to 10,000 microneedles per 1 cm ^2, and at least one kind disposed on the microneedles. This is achieved by a microneedle device for allergy diagnosis provided with allergen antigen holding means for holding allergen antigens.
Here, the microneedle can be produced using a non-metallic synthetic or natural resin material.
The above non-metallic synthetic or natural resin materials are biodegradable polymers such as polylactic acid, polyglycolide, polylactic acid-co-polyglycolide, capronolactone, polyurethane or polyanhydride, or polysaccharides such as hyaluronic acid and pullulan. , Dextran, dextrin or chondroitin sulfate, or polycarbonate, polymethacrylic acid, ethylene vinyl acetate, polytetrafluoroethylene or polyoxymethylene which is a non-degradable polymer.
The allergic antigen can be a peptide or protein.
The allergen-retaining means may be the allergic antigen coated on the surface of the microneedle using a coating carrier.
The allergic antigen can be coated only on the tip of the microneedle surface.
The coating carrier can contain a water-soluble polymer having a hydroxyl group.
The coating carrier may contain a water-soluble polymer having a hydroxyl group selected from the group consisting of polyethylene oxide, polyhydroxymethylcellulose, polyhydroxypropylcellulose, polyhydroxypropylmethylcellulose, polymethylcellulose, dextran, polyethylene glycol, and polyvinyl alcohol. it can.
The coating carrier can contain a polysaccharide. The polysaccharide may contain a water-soluble polymer selected from the group consisting of pullulan, carmellose sodium, chondroitin sulfate, hyaluronic acid, dextran, and gum arabic.
The coating can be fixed to the surface of the microneedle.

  The allergic patch test, which normally took 48 hours, has just made it possible to make a clear skin determination in a short time, for example, about 20 minutes, and with a simple operation using the diagnostic microneedle device of the present invention. Therefore, it is possible to provide a cheaper allergy diagnosis microneedle device that is less painful and substantially avoids bleeding.

It is a figure which shows an example of the microneedle device for allergy diagnosis which concerns on this invention, (a) is a perspective view, (b) is AB sectional drawing of (a). It is a graph which shows the leakage amount evaluation result of Evans blue.

1A and 1B are diagrams showing an example of a microneedle device for allergy diagnosis according to the present invention, in which FIG. 1A is a perspective view and FIG. 1B is a cross-sectional view taken along line AB of FIG. As shown in FIG. 1 (a), a microneedle device 1 according to the present invention includes a microneedle substrate 2, and a plurality of microneedles 3 arranged in a two-dimensional shape capable of perforating skin on the microneedle substrate 2. Have On the microneedle 3, a coating 4 is applied using a coating carrier as an allergy antigen holding means for holding at least one type of allergen. The coating 4 is preferably fixed to the surface of the microneedle 3. Although not shown, the coating 4 may be provided on the microneedle substrate 2 on which the microneedles 3 are provided. The microneedle 3 is preferably made using a non-metallic synthetic or natural resin material. Moreover, although the shape of the microneedle 3 is a conical shape in this example, the present invention is not limited to this, and may be a polygonal pyramid such as a quadrangular pyramid or another shape.
The microneedle device includes a needle portion (microneedle) punctured into the skin or mucous membrane and a substrate that supports the needle portion, and a plurality of needle-like structures are arranged on the substrate. In the present invention, since the needle-like structure of the microneedle handles a minute structure, the length (height h) of the microneedle is preferably 50 μm to 500 μm. Here, the length of the microneedle is set to 50 μm or more in order to ensure administration of the allergic antigen through the skin, and the length of 500 μm or less is used to avoid contact with the nerve of the microneedle and to reduce pain. This is because the possibility can be surely reduced and at the same time the possibility of bleeding is surely avoided. Further, when the length is 500 μm or less, the amount of the antigen that enters the skin can be efficiently administered.

Here, the microneedle means a convex structure and means a needle shape in a broad sense or a structure including a needle shape. In the case of a conical structure, the diameter at the bottom is usually about 50 to 200 μm. is there.
In addition, since the shape is not limited to a simple needle shape, and there is a shape having no sharp point, the microneedle here is not limited to a shape having a sharp tip.

  The microneedle substrate is a base for supporting the microneedle, and the form thereof is not limited. For example, the microneedle substrate may be a substrate having a through-hole, thereby allowing antigen administration from the back surface of the substrate. It becomes possible. Examples of the material of the microneedle or the substrate include silicon, silicon dioxide, ceramic, metal (stainless steel, titanium, nickel, molybdenum, chromium, cobalt, etc.) and synthetic or natural resin materials. Considering the unit price of the material, biodegradable polymers such as polylactic acid, polyglycolide, polylactic acid-co-polyglycolide, pullulan, capronolactone, polyurethane, polyanhydride, polycarbonates that are non-degradable polymers, polymethacrylic acid, A synthetic or natural resin material such as ethylene vinyl acetate, polytetrafluoroethylene, or polyoxymethylene is particularly preferable. Also suitable are polysaccharides such as hyaluronic acid, pullulan, dextran, dextrin or chondroitin sulfate.

The density of the microneedles is typically spaced between the rows such that the rows of needles provide a density of about 1 to 10 per millimeter (mm). In general, the rows are separated by a substantially equal distance with respect to the space of the needles in the row and have a needle density of 100 to 10,000 per cm ² . When the needle density is 100 or more, the amount of antigen entering the skin can be efficiently administered, and when the needle density exceeds 10,000, it is difficult to give the microneedles strength capable of perforating the skin.

  Microneedle manufacturing methods include wet etching or dry etching using a silicon substrate, precision machining using metal or resin (electric discharge machining, laser machining, dicing, hot embossing, injection molding, etc.), machinery Examples include cutting. By these processing methods, the needle portion and the support portion are integrally molded. As a method for making the needle part hollow, there is a method of performing secondary processing by laser processing or the like after producing the needle part.

  Further, in the present invention, the antigen can be coated on the microneedle using purified water and / or a polymer coating carrier or purified water and / or a low molecular weight coating carrier. Higher permeability can be secured even in a compound. That is, polyethylene oxide, polyhydroxymethylcellulose, polyhydroxypropylcellulose, polyhydroxypropylmethylcellulose, polymethylcellulose, dextran, polyethylene glycol, polyvinyl alcohol as a polymer coating carrier, and salts such as sodium chloride and glucose as a low molecular weight coating carrier A coating agent selected from saccharides is coated on the entire surface or part of the microneedles and then dried. Upon application, the coated microneedle punctures the stratum corneum of the skin, so that antigen from the coating is percutaneously absorbed through the perforated hole. In this case, the coating can be dissolved with body fluid from the skin, but a liquid for dissolving the coating may be separately applied to the application part of the skin or the coating itself.

The coating carrier is preferably a polymer coating carrier, of which polyethylene oxide, polyhydroxymethyl cellulose, polyhydroxypropyl cellulose, polyhydroxypropyl methyl cellulose, polymethyl cellulose, dextran, polyethylene glycol, polyvinyl alcohol, pullulan, which are water-soluble polymers having a hydroxyl group. Carmellose sodium, chondroitin sulfate, hyaluronic acid, gum arabic and the like are more preferable. Particularly preferred is polyvinyl alcohol. The saponification degree of polyvinyl alcohol is 78 to 100 mol%, and a grade having a relatively low saponification degree is particularly preferable. In the case of a partial or intermediate saponification grade such as PVA-220 (manufactured by Kuraray Co., Ltd.), the saponification degree is 94 mol. %. Moreover, the average degree of polymerization of polyvinyl alcohol is 200 to 5000, and 500 to 2000, which tends to have relatively high solubility, is most effective.
In consideration of compatibility with the allergen having a relatively large molecular weight (property of uniformly intersecting), a polysaccharide carrier is preferable, and polyhydroxymethylcellulose, hydroxypropylcellulose, polyhydroxypropylmethylcellulose, polymethylcellulose, dextran is preferable. Polyethylene glycol, pullulan, sodium carmellose, chondroitin sulfate, hyaluronic acid, dextran, gum arabic and the like are preferred, and hydroxypropyl cellulose, pullulan, gum arabic are more preferred. In particular, hydroxypropyl cellulose (HPC-SSL (molecular weight: 15,000 to 30,000), HPC-SL (molecular weight: 30,000 to 50,000), HPC-L (molecular weight: 55,000 to 70,000) HPC-M (molecular weight: 110,000 to 150,000), HPC-H (molecular weight: 250,000 to 400,000)), pullulan and hyaluronic acid are most preferred.

  The content of the coating carrier in the coating agent is 1 to 70% by weight, preferably 1 to 40% by weight, particularly preferably 3 to 25% by weight. In addition, this coating carrier may require a certain degree of viscosity so that it does not drip, and the viscosity is required to be about 100 to 100,000 cps. A more preferred viscosity is 500 to 50000 cps, and a most preferred viscosity is 5000 to 30000 cps.

  Also, the coating thickness on the microneedles is less than 50 μm, and most preferably less than 25 μm, ie 1 to 10 μm. In general, the coating thickness is the average thickness measured across the surface of the microneedle after drying. The thickness of the coating can generally be increased by adapting multiple coatings of the coating carrier and can be formed by drying the coating between successive coatings. The coating is applied to the microneedles using a known method and is fixed by drying. It is also possible to apply to the inner surface of the hollow path of the needle-like structure of the microneedle and the lower surface of the substrate of the microneedle. It is sufficient if the coating is applied only to the tip.

  The liquid composition used to coat the microneedles is prepared by mixing the biocompatible carrier, the beneficial agent to be delivered, and possibly any coating aids with the volatile liquid. To do. The volatile liquid can be water, dimethyl sulfoxide, dimethylformamide, ethanol, isopropyl alcohol and mixtures thereof. Of these, water is most preferred. The liquid coating solution or suspension can typically have a beneficial bioactive substance concentration of 0.1-65% by weight, preferably 1-30% by weight, more preferably 3-20%. % By weight. The coating is particularly preferably in a fixed state. The “fixed state” refers to a state in which the coating carrier is substantially uniformly attached to the object. However, immediately after coating, the coating carrier is fixed in a dry state by a known drying method such as air drying, vacuum drying, freeze drying, or a combination thereof, but after transdermal administration, moisture in equilibrium with the surrounding atmosphere. Since the content or the organic solvent may be retained, it is not always fixed in a dry state.

  Other known formulation aids may be added to the coating as long as they do not deleteriously affect the required solubility and viscosity characteristics of the coating and the physical integrity of the dried coating.

In the case of allergy tests for metals and chemical substances, the corresponding metals and chemical substances are used as allergic antigens. In the case of allergy tests to clarify antigens for atopic dermatitis, house dust, mites, fungi, Components such as bacteria and food can be used. Furthermore, in pollinosis, various pollens such as cedar, cypress, ragweed, mugwort, birch, rice are used. Moreover, the antigen of a tuberculin reaction can also be used.
For food allergies, various food constituents such as eggs, milk, dairy products, meat, rice grains, beans, fish, and fruits that are said to be involved in the onset and exacerbation can be used.

  These components can be used as crude products extracted by general techniques, or as purified single components, and are produced in large quantities by incorporating genes into microorganisms and animal cells using genetic engineering. You can also use it. Furthermore, degradation products of these antigens, arbitrary fragment peptides, or those obtained by mutating some amino acids can be used. In addition, these antigens can be used alone, but by using two or more combinations, a method of use expecting a wider effect can be used.

(Experimental example 1)
Anaphylactic model rats were obtained in which male lysozyme / complete Freund's adjuvant (1: 1) was intraperitoneally administered (400 μg / rat) to male 12 W hairless rats, and immunity to protein lysozyme (IgE) was acquired after 12 days. .
Next, under urethane anesthesia, 3% Evans blue (EB) solution (100 μL / per 100 g body weight) was administered into the jugular vein, and protein lysozyme was shown on the left and right sides of the abdomen as shown in Comparative Examples 2 to 4 and Examples 1 and 2 below. Administered. In Comparative Example 1, protein lysozyme is not administered.
The evaluation of coloring with Evans Blue was performed as follows. That is, the skin at the administration site was peeled off, punched out with a skin punch having an inner diameter of 12 mm (φ12), the skin section was removed, and the skin section was melted with 1 mL of 1N potassium hydroxide and allowed to stand at 37 ° C. overnight. The dissolved skin section is added with 2.5 mL of 0.6N phosphoric acid solution and 6.5 mL of acetone, centrifuged (1100 * g, 20 minutes), and 200 μL of the supernatant is collected with a plate reader (spectrophotometer). It was measured. FIG. 2 shows the evaluation result of the leakage amount of Evans Blue.

(Comparative Example 1)
Comparative Example 1 was obtained by dropping 20 μL of saline without administering protein lysozyme in the operation of Experimental Example 1.

(Comparative Example 2)
When administering protein lysozyme in the operation of Experimental Example 1, a microneedle (made of polylactic acid, length 250 μm, density 841 / cm ² ) without any coating was punctured for 5 seconds, and then 5% protein lysozyme. A non-woven fabric preparation (area 1 cm ² ) impregnated with the solution (400 μL) was applied for 10 minutes.

(Comparative Example 3)
During the intradermal administration of protein lysozyme in the procedure of Experimental Example 1, 20 μL of 5% protein lysozyme solution was dropped onto a microneedle (made of polylactic acid, length 250 μm, density 841 / cm ² ) that was not coated with anything. Then, the microneedle device was stuck for 10 minutes after pressing.

(Comparative Example 4)
Of the operations in Experimental Example 1, 25 μL of protein lysozyme 0.5% was intradermally administered as Comparative Example 4.

Example 1
It was dissolved in a 15% solution of polyvinyl alcohol (PVA220: Kuraray Co., Ltd.) so that the protein lysozyme concentration would be 5%, and microneedles (made of polylactic acid, length: 250 μm, density: 841) three times using a metal mask / Cm ² ) and dried. Next, this microneedle device was stuck for 10 minutes after pressing.

(Example 2)
Example 1 was dissolved in Example 1 so that the protein lysozyme concentration was 10%.
In the graph of FIG. 2 showing the leakage amount evaluation result of Evans Blue, the value of Comparative Example 1 is indicated by a horizontal line. As can be seen from the graph in FIG. 2, the Evans blue coloring is clearly higher in Examples 1 and 2 than in the comparative example. Thus, it was found that administration of protein lysozyme (antigen) by coating microneedles has caused a more effective allergic reaction.

(Example 3) Compatibility check test operation method of various polymers with BSA and OVA Various aqueous solutions and BSA and OVA were prepared according to the conditions in Tables 1 and 2 below, respectively, and mixed aqueous solutions were prepared. Compatibility was evaluated by confirming the presence or absence of occurrence and the occurrence of phase separation after centrifugal defoaming (centrifugal conditions are listed in the table) (uniform liquidity: ○ mark, non-uniform liquidity: x mark) ). In Tables 1 and 2, the ◯ marks are compatible, and the X marks are not compatible. In the following description,% notation is weight%. The coating content was measured by the method shown in FIG. 2 described above, followed by extraction with 1 mL purified water, and the BSA or OVA content (adhesion amount) was measured. Moreover, the description of “impossible” indicates that adhesion of the polymer to the needle was not recognized.

  Tables 1 and 2 show the results regarding the compatibility of OVA or BSA with each water-soluble polymer and the BSA or OVA content when the microneedle is coated. Pullulan, hydroxypropylcellulose (HPC), methylcellulose, hyaluronic acid, and sodium polyacrylate showed high compatibility by optimizing the blending ratio of bioactive substance and water-soluble polymer. High compatibility with OVA concentration. Furthermore, as a result of coating the microneedles by the method of FIG. 2 using these solutions, pullulan showed the highest value, followed by hydroxypropylcellulose (SL), methylcellulose, and hyaluronic acid in this order. Regarding hydroxypropylcellulose, a difference in coating amount was recognized depending on the grade, and a tendency to decrease in the order of HPC-SL> HPC-L> HPC-H was shown. This is probably because hydroxypropylcellulose showed a tendency for the viscoelasticity (viscosity) of the polymer to increase as the molecular weight decreased, and thus the adhesion to the microneedles increased. In addition, methyl cellulose showed good compatibility with OVA, but good conditions were not confirmed with BSA. As for hyaluronic acid, both OVA and BSA showed good compatibility, but the viscosity was poor and a sufficient coating amount could not be obtained. Polyacrylic acid Na showed good compatibility, but no adhesion to the needle was observed, making it unsuitable as a coating carrier. From the above results, by using a coating carrier having compatibility with the polymeric physiologically active substance, a coating containing a substantially uniform polymeric physiologically active substance can be realized.

  The following polymers were used. Methylcellulose (SM-25, SM-400, SM-8000) is manufactured by Shin-Etsu Chemical Co., Ltd., polyacrylic acid (NP-600, NP-800) is manufactured by Showa Denko, and hydroxypropylmethylcellulose (90SH-30000, 65SH-1500). , TC-5) manufactured by Shin-Etsu Chemical Co., Ltd., and polyvinylpyrrolidone (K29 / 32, K90) manufactured by Nippon Shokubai Co., Ltd. were used.

  As described above, according to the present invention, not only can the skin be determined more clearly in a short time and with a simple operation, but also a more inexpensive microneedle for allergy diagnosis can be provided. It greatly contributes to the development of industry and related industries.

Claims (7)

A substrate, a length of 50 μm to 500 μm that is provided on the substrate and capable of perforating the skin, has a density of 100 to 10,000 microneedles per 1 cm ^2, and is disposed on the microneedles to hold at least one allergen. Bei example the allergen holding means,
The microneedle is made using polylactic acid,
The allergen holding means is coated on the surface of the microneedle with the allergen using a coating carrier,
A microneedle device for allergy diagnosis , wherein the coating carrier contains pullulan, hydroxypropylcellulose, methylcellulose, hyaluronic acid or a polyacrylic acid partial neutralized product . The microneedle device for allergy diagnosis according to claim 1, wherein the allergic antigen is a peptide or a protein. 3. The microneedle device for allergy diagnosis according to claim 1 or 2 , wherein the coating is in a state of being fixed to the surface of the microneedle.   The microneedle device for allergy diagnosis according to any one of claims 1 to 3, wherein when pullulan is used as the coating carrier, the polymer concentration is 15% or more.   4. When using hydroxypropyl cellulose as the coating carrier, hydroxypropyl cellulose-SL (molecular weight: 30,000 to 50,000) is used at a polymer concentration of 25% or more. A microneedle device for allergy diagnosis as described in 1.   When using hyaluronic acid as the coating carrier, hyaluronic acid (molecular weight: 900,000) is used at a polymer concentration of 1% or more, for allergy diagnosis according to any one of claims 1 to 3. Microneedle device.   The microneedle device for allergy diagnosis according to any one of claims 1 to 3, wherein when the partially neutralized polyacrylic acid is used as the coating carrier, the polymer concentration is 1.5% or more. .

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